This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally capped and with the C‐terminus adapted to give an electrophilic trap. Such compounds are also at the basis of the research described in this Thesis. Attention was directed to substitute specific amino acid residues by either synthetic, non‐canonical amino acid derivatives (with a review on the synthesis of such items given in Chapter 2) or dipeptide isosteres. Alternatively, modifications of the electrophilic trap, specifically, the epoxyketone moiety, were investigated. In this way, and by the synthesis of focused libraries, in which in each case a number of structural analogues, rather than a single one, inhibitors selective for β5c, β2c and β2i were discovered, and a number of two‐step activity‐based probes for probing these activities in vitro and in situ were identified.